The acetylcholine (ACh) receptors of electric tissues and of skeletal muscle transduce the binding of ACh into an increase in the permeability of the post-synaptic membrane to cations. The receptor is an integral membrane protein. Receptor isolated from Torpedo californica has a chain composition of A2BCD. The A chains contain the ACh binding sites. We will tag three of the four types of chains for electron microscopic identification and will determine the relative positions of the tagged chains in negatively stained and in freeze-dried receptor. Using peptide mapping, we will characterize and order a set of proteolytic fragments of each chain. We will determine the accessibility of the chains in native receptor-rich membrane to impermeant reagents and proteases and to hydrophobic reagents and will locate the accessible sites on the proteolytic fragments. We will thus determine the arrangement of the chains in the direction normal to the plane of the membrane. Functional sites in or near the cation channel will be covalently modified with fluorescent and photogenerated labels. Both the influence of the functional state of the receptor on the labeling reaction and the effect of labeling on the function of the receptor will be determined. The functional sites so identified and the ACh binding sites, affinity labeled as we previously described, will be located at the level of the proteolytic fragments and, thus, in the three dimensional framework of the receptor chains in the membrane. The establishment of the spatial relationships of the functional sites will provide a basis for our understanding the function of the receptor in terms of its molecular structure.